细胞角蛋白19片段抗原21-1含量化学发光微粒子定量免疫检测方法的建立

Development of chemiluminescent microparticle immunoassay for cytokeratin 19 fragment 21-1 content

目的建立细胞角蛋白19片段抗原21-1(cytokeratin 19 fragment 21-1,CYFRA21-1,简称CY21-1)含量的化学发光微粒子免疫检测方法(chemiluminescent microparticle immunoassay,CMIA)。方法原核表达制备重组CY21-1(r CY21-1),用其免疫BALB/c小鼠后,收集小鼠脾脏细胞,常规杂交瘤技术与Sp2/0进行融合,制备CY21-1单抗。间接ELISA法筛选可稳定分泌CY21-1抗体的杂交瘤细胞,分别标记微性磁珠(magnetic beads,MB)和吖啶酯(acridinium ester,AE),筛选可特异检测CY21-1的单抗配对,建立CY21-1 CMIA,同时验证方法的线性及灵敏度,并与同类试剂盒进行比较。结果经筛选获得了一组能特异检测CY21-1的单抗配对MB*26B5-3E4*AE,建立的CY21-1 CMIA对检测CY21-1校准品的线性范围为0.1~1 000 ng/m L,R^2=0.992 3,检测灵敏度为0.076 ng/m L。该方法与美国雅培公司生产的Abbott CY21-1 CMIA诊断试剂盒平行检测250份临床血清标本的结果具有良好的相关性(R^2=0.961 6)。结论成功建立了CY21-1 CMIA,且具有良好的线性及灵敏度,为我国肺癌的临床筛查和早期诊断奠定了基础。

Objective To develop a chemiluminescent microparticle immunoassay（CMIA） for cytokeratin 19 fragment21-1（CYFRA21-1,CY21-1 for short） content. Methods Recombinant CY21-1 antigen was prepared by prokaryotic expression and used for immunization of BALB/c mice. The splenocytes of mice were collected and fused with Sp2/0 cells by routine hybridoma technique. The hybridoma cells stably secreting the monoclonal antibody（m Ab）against CY21-1 were screened by indirect ELISA and labeled with magnetic beads（MB） and acridinium ester（AE） respectively. The m Ab pair for specific detection of CY21-1 were screened,based on which CY21-1 CMIA was developed,verified for linearity and sensitivity,and compared with the kits of same kind. Results An antibody pair MB＊26 B5-3 E4＊AE was obtained,with which CY21-1 CMIA was developed. The linear range of the developed method for determination of CY21-1 calibrator was 0. 1-1 000 ng/m L,with a R^2 value of 0. 992 3,while the sensitivity was 0. 076 ng/m L. The determination results of 250 human serum samples by the developed method showed good correlation with that by commercial Abbott CY21-1 CMIA kit（R^2 = 0. 961 6）. Conclusion CY21-1 CMIA was developed successfully,which showed high linearity and sensitivity. It laid a foundation of clinical screening and early diagnosis of lung cancer in China.